The post-transcriptional acquisition of a poly(A) tail onto the 3' ends of eukaryotic mRNAs is an essential process that promotes transcription termination and transport of mRNA from the nucleus and serves as an additional point at which the cell can regulate the type and amount of mRNA derived from a particular gene. The poly(A) tail is also important for optimal translation and for determining mRNA stability. Polyadenylation requires site-specific endonucleolytic cleavage of the primary transcript followed by poly(A) addition to the upstream product. These steps are closely coupled in vivo, but can be experimentally uncoupled in vitro and assayed separately. While many of the protein components required for each step have been identified, much less is known about the biochemical nature of the process. Using the yeast S. cerevisiae as a model eukaryote, we will address the following specific aims: 1. Analysis of the molecular mechanism by which yeast cleavage factors recognize and cleave the mRNA precursor in the 3' untranslated region. These experiments will determine what constitutes the core cleavage complex, and how these factors interact with each other and with the RNA to mediate this step of the reaction. 2. Investigation of the molecular linkage between the mRNA 3'-end processing machinery and the mRNA transport apparatus. This aim seeks to identify connections between disassembly of the polyadenylation complex and assembly of the transport complex. We will ask when the cleavage/polyadenylation factors leave the polyadenylated product, and whether this recycling is facilitated by transport factors. A closely related issue is when transport factors join the mRNA, and whether their recruitment is assisted by the polyadenylation factors. With the research proposed here, we hope to derive a dynamic model of the complex that cleaves mRNA precursor. This work should provide insight into how this complex sets the stage for subsequent polyadenylation and transport of the mRNA. A better understanding of the basic mechanism will also make it easier to determine how polyadenylation is coordinated with other nuclear events and how the cellular environment modulates the activity or levels of the factors involved in this essential process.